TY - JOUR
T1 - C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity
AU - Murillo-de-Ozores, Adrián Rafael
AU - Rodríguez-Gama, Alejandro
AU - Bazúa-Valenti, Silvana
AU - Leyva-Ríos, Karla
AU - Vázquez, Norma
AU - Pacheco-Álvarez, Diana
AU - De La Rosa-Velázquez, Inti A.
AU - Wengi, Agnieszka
AU - Stone, Kathryn L.
AU - Zhang, Junhui
AU - Loffing, Johannes
AU - Lifton, Richard P.
AU - Yang, Chao Ling
AU - Ellison, David H.
AU - Gamba, Gerardo
AU - Castañeda-Bueno, Maria
N1 - Publisher Copyright:
© 2018 American Society for Biochemistry and Molecular Biology Inc. All rights reserved.
PY - 2018/8/3
Y1 - 2018/8/3
N2 - WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.
AB - WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.
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U2 - 10.1074/jbc.RA118.003037
DO - 10.1074/jbc.RA118.003037
M3 - Article
C2 - 29921588
AN - SCOPUS:85051119240
SN - 0021-9258
VL - 293
SP - 12209
EP - 12221
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 31
ER -